The present invention is directed to the discovery of antibodies that will react to proteins or nucleic acids bound to particular crosslinkers, but not to the free crosslinkers or free proteins or nucleic acids. Monoclonal antibodies with such binding specificity have widespread applications in receptor-ligand binding, immunodiagnostic, molecular amplification assays and other nucleic acid diagnostics.
DSS (Dissuccinimidyl suberate) is a non-cleavable, membrane permeable, amine-reactive, homobifunctional crosslinker. BS3 (Bis[Sulfosuccinimidyl]suberate) is a non-cleavable, membrane impregnable, water soluble analog of DSS. By means of two bifunctional sulfo-NHS ester reactive groups, BS3 (and DSS) can serve as a crosslinking reagent between molecules with amino groups. The BS3 and DSS compounds are sold by Pierce (Rockford, Ill.) as protein crosslinking reagents. These compounds will crosslink molecules that are within a certain distance (i.e. spacer arm length), but otherwise will modify such molecules without necessarily crosslinking them. Thus, BS3 and DSS can be used as labels of analytes.
Such modifications of proteins and nucleic acids by digoxigenin, for instance, are known in the art. In general, digoxigenin has been used as a label in bioanalytical assays where it may be itself radiolabelled or may act as a hapten, for instance, which reacts with an anti-hapten antibody for detection of the digoxigenin-labeled analyte. See U.S. Pat. Nos. 3,855,208; 5,198,537; and 5,804,371.
Digoxigenin and derivatives thereof have also been used in the field of nucleic acid diagnostics, where in general it is incorporated as a label into amplificates or probes, and whereby the labelled moieties are detected by hapten anti-hapten reaction principle. See, for example, U.S. Pat. Nos. 5,354,657; 5,843,670; 5,929,108; and 5,344,757.
However, there are drawbacks of the digoxigenin system. First, the procedure for derivatizing with digoxigenin is relatively complicated. Second, because digoxigenin is a large molecule and contains a hydrophobic steroid, modification of a molecule will perturb the molecule""s conformation. Third, digoxigenin is relatively expensive, as compared to for instance BS3.
Therefore, a need exists for a simpler bioanalytical detection system, which does not require multiple derivatization steps, is less expensive, and is less likely to disrupt the conformation of the molecule being modified therewith. A system which is parallel in many respects to the digoxigenin system has now been, unexpectedly, discovered. This system can be used in the same manner as digoxigenin.
The present invention is the result of the discovery that certain monoclonal antibodies produced by hybridomas raised against BS3-modified gp120-CD4 complexes were actually directed to the BS3 linker itself. These antibodies do not react with the free BS3 molecule alone, and show different binding specificities. For instance, some of the antibodies appear to react with the xe2x80x9chingexe2x80x9d formed between amino acid residues and the BS3 molecule in a modified protein. These antibodies would be expected to crossreact with proteins treated with other crosslinkers, such as DSG (Pierce) and DTSSP (a molecule analogous to DSS but with an Sxe2x80x94S bridge in the middle of the methylene chain). Other monoclonals react with the linear carbon chain that lies between the two end sulfosuccinimidyl groups of the BS3 molecule. Thus, such monoclonals would also be expected to react with DSS modified (or crosslinked) proteins, because it contains the same long methylene chain, and presumably other crosslinkers such as DMP, DMA, DSG and MSA (all sold by Pierce).
The monoclonals of the present invention are useful in diagnostic immunoassays, such as ELISAs. They are also useful in ligand-receptor studies. Finally, it is also contemplated that the BS3anti-BS3 system can be used as a detection system for nucleic acid amplification assays. In fact, these anti-hapten antibodies can be used in the same manner as other hapten/anti-hapten systems known in the art, such as digoxigenin/anti-digoxigenin.